A Novel snoRNA, Gm24418 Attenuates Inflammation Injury After Acute TBI Through Regulating CCL2

Introduction

Traumatic brain injury (TBI), caused by external mechanical forces disrupting normal brain function, is a significant health issue on a global scale.¹ It is estimated that TBI contributes to approximately 50–60 million cases annually and imposes a cumulative economic cost of $400 billion worldwide.² Accumulating evidence indicates that complex pathophysiological processes—including neuroinflammation,³ cerebral edema, and other secondary injuries^4,5 —during the transition from acute to chronic stages of TBI contribute progressively to persistent neurological deficits and elevated mortality risk.⁶ Therefore, early diagnosis and timely intervention are critical for mitigating disease progression and improving patient outcomes. Notably, neuroinflammation has been increasingly recognized as a central mechanism driving long-term neurological sequelae, correlating with worse cognitive outcomes and increasing mortality in TBI patients.^7,8

Substantial heterogeneity exists among individuals with respect to both injury severity and recovery patterns.^9,10 Conventional imaging techniques, including Computed Tomography (CT) and Magnetic Resonance Imaging (MRI), often lack the sensitivity to detect subtle or diffuse neuronal damage, particularly in TBI.^11,12 This diagnostic limitation highlights the pressing need for reliable molecular biomarkers capable of accurately reflecting the extent of brain injury and predicting clinical trajectories.^13,14

Small nucleolar RNAs (snoRNAs)—evolutionarily conserved non-coding RNAs—direct pseudouridylation (H/ACA-box),¹⁵ and site-specific 2′-O-methylation (C/D-box) of ribosomal RNA,¹⁶ while also regulating mRNA splicing and ribosome biogenesis through sequence-specific interactions. Beyond their typical roles in rRNA modification, an increasing number of “orphan” snoRNAs are being associated with non-standard functions,¹⁷ such as the regulation of alternative splicing,^18,19 responses to cellular stress, and chromatin remodeling.²⁰ Several snoRNAs, including SNORD115 and SNORD116, are highly enriched in the central nervous system and have been shown to modulate splicing of the serotonin receptor 5-HT2C and influence fear-related memory consolidation.²¹ Furthermore, aberrant expression of snoRNAs has been reported in various neurological disorders, such as autism spectrum disorder and amyotrophic lateral sclerosis.²²

Recent research indicates that long non-coding RNAs (lncRNAs) are involved in modulating neuroinflammatory pathways associated with TBI.²³ Additionally, snoRNAs, a distinct class of conserved non-coding RNAs primarily involved in ribosomal RNA modification, have recently been recognized as critical yet underappreciated contributors to the molecular landscape of TBI. Emerging evidence suggests that snoRNAs play a role in the pathophysiological response to TBI. Preliminary human studies have identified specific changes in circulating snoRNAs following injury. For example, Ho et al²⁴ reported that four snoRNAs (ACA48, U35, U55, and U83A) are significantly downregulated in peripheral blood mononuclear cells (PBMCs) from veterans with comorbid mild TBI (mTBI) and post-traumatic stress disorder (PTSD) compared to those from PTSD-only controls, indicating their potential as diagnostic biomarkers. In comparison to conventional protein-based biomarkers, snoRNAs exhibit enhanced stability in biofluids,²⁵ primarily due to their encapsulation within extracellular vesicles or their association with RNA-binding proteins such as Ago2, which provides resistance to RNase degradation.^26,27 Their enrichment in brain-specific regions and rapid expression changes following injury highlight their potential as sensitive and specific biomarkers for the diagnosis and prognosis of TBI.^28,29 The role of snoRNAs in central nervous system (CNS) injuries is gaining increasing recognition; a recent comprehensive review underscores the emerging utility of ncRNAs, including snoRNAs, as diagnostic and therapeutic biomarkers for conditions such as TBI.³⁰ Although these findings are promising, the functional implications of altered snoRNA expression within the injured brain parenchyma remain largely unexplored. Consequently, investigating the cell-type-specific expression and functional roles of snoRNAs directly in the brain is a critical next step in understanding their contribution to TBI pathology and in identifying novel therapeutic targets.³¹

In our research, we used small RNA microarray and bioinformatics analysis to detect snoRNAs with differential expression in mice with TBI. We examined the downstream target genes and signaling pathways linked to Gm24418 to clarify the possible mechanisms behind its impact on inflammatory responses in the peripheral and central nervous systems of TBI mice.

Materials and Methods

Ethics Statement

All mouse experiments were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC)of Central South University (Ethical Approval No. CSU-2023-0386). All procedures adhered to the Central South University guide for the Care and Use of Laboratory Animals and prioritized the 3Rs principle (Replacement, Reduction, Refinement) to minimize animal suffering. All animal experiments were conducted strictly in alliance with ARRIVE guidelines.

Experimental Animals

Male C57BL/6J mice (6–8 weeks old, 20–28 g) were sourced from Central South University Laboratory Animal Center. Animals were housed in specific pathogen-free (SPF) facilitiesunder a 12-h light/dark cycle (lights on: 08:00–20:00) at 21 ± 1.5°C and 50 ± 10% humidity. Mice underwent a 7-day acclimation periodprior to experimentation to minimize environmental stress.

Establishment of the TBI Model

A standardized controlled cortical impact (CCI) protocol was implemented to induce reproducible TBI in C57BL/6 mice. The experimental animals were randomly assigned to two groups—Sham and CCI (n = 30 per group)—using an online random number generator. Sample size was determined based on prior studies, and all eligible animals were included with appropriate controls to maximize statistical power while minimizing the number of animals used, in accordance with the ARRIVE guidelines. Anesthesia was induced via intraperitoneal injection of 0.3% pentobarbital sodium (60 mg/kg), followed by verification of surgical tolerance through reflex monitoring. Mice were secured in a stereotaxic frame, and a 4 mm craniotomy was performed over the right parietal cortex (coordinates: 2.0 mm lateral to bregma, 1.5 mm posterior to lambda). The CCI device was calibrated to deliver cortical deformation at 1.5 mm depth, 5 m/s velocity, and 100 ms duration, ensuring consistent injury severity. All procedures were conducted under aseptic conditions with thermal regulation (37 °C warming pad). Sham controls underwent identical anesthesia and craniotomy without cortical impact. Post-surgery, the surgical site was irrigated with sterile saline and closed with interrupted sutures. Mice were monitored on a heating pad until full recovery of motor function and consciousness.

Animal Dissection and Tissue Collection

All mice were humanely euthanized via intraperitoneal administration of an overdose of sodium pentobarbital at 3 days post-modeling (60 mg/kg, i.p.; Sigma), with post-euthanasia confirmation of irreversible death achieved using AVMA-recommended criteria, including absence of heartbeat, respiration, pupillary light reflex, and corneal reflex. Transcardial perfusion with ice-cold saline was conducted only after verification of death to ensure optimal tissue preservation. For morphological analysis, brains were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned coronally at a thickness of 3 μm (n = 5 per group). For molecular profiling, including snoRNA-seq, RNA-seq, and qRT-PCR, the cortical region immediately surrounding the injury site (perilesional zone) from the injured hemisphere were rapidly dissected, immediately snap-frozen in liquid nitrogen, and stored at –80 °C (n = 5 per group). All experimental procedures were performed in strict accordance with the AVMA Guidelines for the Euthanasia of Animals (2020) and were approved by the Institutional Animal Care and Use Committee.

RNA Extraction

Total RNA was extracted from samples using the TRIzol reagent (Invitrogen) following standard molecular biology protocols. Concentrations of RNA were determined with a NanoDrop ND-1000 spectrophotometer, and RNA integrity was verified through an Agilent 2100 Bioanalyzer or by running aliquots on denaturing agarose gels to assess degradation.

RNA Labeling

For microarray analysis of small RNAs, 100 ng total RNA underwent dephosphorylation with T4 polynucleotide kinase (3 units, 37°C, 40 min) to remove 3′ phosphorylation. DMSO (7 μL) was added for denaturation (100°C, 3 min), followed by rapid cooling. Cy3-labeled pCp was incorporated using T4 RNA ligase (15 units, 16°C, overnight) in a 28 μL reaction containing ligase buffer and BSA.

Array Hybridization

The labeled product was combined with 2× hybridization buffer (Agilent) to reach a final volume of 45 μL. This mixture was briefly heated at 100 °C for 5 minutes and cooled on ice prior to loading onto the microarray slides. Hybridization was carried out at 55 °C for 20 hours. Post-hybridization washing included an initial rinse in 6 × SSC containing 0.005% Triton X-102 for 10 minutes at room temperature, followed by a second wash in 0.1× SSC with the same detergent concentration for 5 minutes. The microarrays were scanned using an Agilent G2505C platform to obtain fluorescence intensity images.

Behavioral Testing

Neurological function was appraised using a modified neurological severity score (mNSS), which quantifies sensorimotor coordination and postural balance. Higher scores correspond to greater neurological impairment. Motor coordination deficits were further evaluated by a foot-fault assay performed on an elevated wire grid measuring 80 cm², composed of 25 mm square meshes. Mice were placed individually on the grid and allowed to explore for one minute while behavior was video recorded. The proportion of contralateral missteps relative to total steps was calculated using the formula: [(contralateral faults - ipsilateral faults) / (total steps)] × 100%. Body weight alterations were computed as deviations from baseline measurements obtained before injury induction. All testing was executed by a blinded investigator at days 1 and 3 post-modeling (n = 5 per group).

Histological Staining

Paraffin sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Hematoxylin–eosin (H&E) staining (Servicebio, Wuhan, China) and Nissl’s staining (Servicebio, Wuhan, China) were performed following standard histological protocols. Images were captured using an Axio Imager M2 microscope (Carl Zeiss, Germany) and analyzed with ImageJ software. Neuronal viability was assessed based on morphological features: viable neurons displayed distinct, large, and lightly stained nuclei, whereas damaged or dying neurons were identified by pyknotic, darkly stained, and shrunken nuclei.

TUNEL Apoptosis Detection

DNA fragmentation was assessed with a commercial TUNEL assay kit (Meilunbio, Dalian, China). Sections were treated with proteinase K for 5 minutes, incubated with TdT enzyme and FITC–12–dUTP at 37 °C for 1 hour, and subsequently washed. Nuclei were counterstained with DAPI (Servicebio, 1:100) for 5 minutes and sealed with anti-fade medium. Images were obtained using a Zeiss fluorescence microscope and processed in ImageJ.

Immunoglobulin G (IgG) Staining

Slides underwent antigen retrieval in citrate buffer (pH 6.0) heated in a microwave for 15 minutes, then cooled to room temperature. After PBS washing, nonspecific binding was blocked with 5% bovine serum albumin (Boster, Wuhan, China) for 1 hour, followed by overnight incubation at 4 °C with anti-mouse IgG conjugated to horseradish peroxidase. Color development was achieved with DAB reagent (Beijing Zhongshan-Golden Bridge, China) for 5 minutes.

Fluorescence in situ Hybridization (FISH) and Immunostaining

FISH detection was performed on cultured neurons and cortical paraffin sections using a Servicebio kit according to manufacturer’s guidelines. Sections were incubated in 3% hydrogen peroxide for 15 minutes to quench endogenous peroxidase activity, followed by blocking with streptavidin in 5% BSA at 37 °C for 30 minutes, and then incubated with 2× SSC for 30 minutes, and hybridized overnight at 37 °C with biotin-labeled probes after denaturation at 75 °C for 10 minutes. Nuclei were counterstained with DAPI and imaged under fluorescence microscopy. For combined FISH–immunostaining, samples were blocked with goat serum and incubated with NeuN antibody (Proteintech, Rosemont, IL, USA) at 4 °C overnight. Secondary antibody incubation was performed for 1 hour, followed by nuclear counterstaining and imaging.

RNA Isolation and qRT-PCR

Total RNA from mouse cortical tissue was extracted using TRIzol (Takara, Beijing, China) and quantified with a NanoDrop One spectrophotometer. Reverse transcription to cDNA was carried out using the HiScript II Q RT SuperMix kit. Quantitative PCR was performed using ChamQ Universal SYBR Master Mix (Vazyme, Nanjing, China). Primer sequences were supplied by Sangon Biotech (Table S1). Relative expression levels were computed with the 2^−ΔΔCt algorithm. snoRNAs were normalized by U6 and mRNAs by GAPDH.

Cell Culture, Viability Testing, and Transfection

N₂A neuroblastoma cells were maintained in high-glucose DMEM supplemented with 10% fetal bovine serum under standard conditions (37 °C, 5% CO₂). Cells (1 × 10⁴ per well) were plated in 96-well plates and treated with lipopolysaccharide (LPS) for 24 hours before viability assessment via CCK-8 assay (Abbkine, Wuhan, China). For transfection experiments, cells (1 × 10⁵ per well) in 12-well plates were transfected with either Gm24418 mimic or negative control using Lipofectamine 3000 (Invitrogen). After 24 hours, fresh medium was introduced, and LPS-treated groups were incubated for an additional 24 hours before RNA collection for qRT-PCR analysis.

Dual-Luciferase Reporter Gene Assay

An analysis of firefly luciferase activity against Renilla luciferase activity was conducted by transfecting HEK293T and N₂A cells with 100 ng of CCL2 plasmid, CCL2-Mut plasmid, and a negative control plasmid, and co-transfecting with 20 ng Renilla plasmid. Forty-eight hours following transfection, a dual luciferase reporter system (Yeasen Biotechnology, China) was employed to assess Luciferase activity.

Adeno-Associated Virus Infection

To increase exogenous expression of Gm24418 in vivo, pAAV-CMV-Gm24418-201-EF1a-EGFP-tWPA (AAV-Gm24418) was used (OBiO, Shanghai, China). Mice were anesthetized with 2% isoflurane and were placed in a stereotactic device. The 10 μL of virus were injected with a 40 mm tip diameter of glass electrode and for more than 10 minutes. 14 days were needed for successful transfection before CCI.

Statistical Analysis

For microarray analysis, raw intensity files were processed via Agilent Feature Extraction v11.0.1.1. Differentially expressed genes(DEGs) were identified using the Limma package in R software. To account for multiple comparisons, p-values were adjusted using the Benjamini-Hochberg method to control the false discovery rate (FDR). Genes with an adjusted p-value (q-value) < 0.05 and |fold-change| > 1.2 were considered statistically significant.

For RNA-seq analysis, raw sequencing reads were quality-checked using FastQC (v0.11.9). Adapter trimming and low-quality read filtering were performed using Trimmomatic (v0.39). Raw counts were normalized using the DESeq2 median-of-ratios method. Differential expression was assessed using the Wald test, and p-values were adjusted for multiple testing using the Benjamini-Hochberg procedure implemented in DESeq2. Genes with an FDR-adjusted p-value (padj) < 0.05 and |log2 fold-change| > 1 were considered differentially expressed.

For experimental assays, values are expressed as mean ± standard error of the mean (SEM). One-way ANOVA for data sets were used with one variable and Two-way ANOVA for data sets were used with two variables, followed by Dunnett’s post hoc test in GraphPad Prism v8. Statistical significance was defined as p< 0.05.

Results

Impairments of TBI on the Brain of Mice

The mNSS and foot fault tests were conducted to evaluate behavioral impairments following traumatic brain injury. On postoperative days 1 and 3, animals subjected to CCI displayed substantially higher mNSS values and foot‑fault percentages compared to sham group (p< 0.0001) (Figure 1A and B). By day 3 post-TBI induction, a notable weight loss was observed in the mice (p< 0.01) (Figure 1C). Brain IgG levels indicate blood-brain barrier permeability; as expected, IgG accumulation was markedly greater in the TBI group than in the sham group (Figure 1D and G). Nissl’s staining allowed for visualization of both the morphological integrity of neurons (Figure 1E and H, black arrow) and damaged neurons (Figure 1E and H, red arrow). At post-injury, histological examination of the damaged cortical region revealed extensive neuronal damaged and morphological degeneration, Apoptotic cells identified via TUNEL assay were rare in sham brains but abundant in the injured tissue (Figure 1F and I).

Figure 1 Neurological deficits and histopathological damage after day 3 CCI. Behavioral assessments reveal significant impairments in CCI mice, as indicated by higher mNSS scores (A) and increased foot faults (B), By day 3 after CCI induction, mice exhibited significant weight loss (C and D) IgG immunoreactivity in brain tissue. (E) Nissl-stained neuronal profiles, with arrows of various colors indicating morphological differences. (F) Representative images of TUNEL staining. (G) Quantification of IgG staining (n = 5). (H) Nissl’s staining quantification (n = 5). (I) Quantification of apoptotic cells detected via TUNEL staining (n = 5). Data are expressed as mean ± SEM. *p <0.05; **p <0.01; ***p <0.001.

Cortical Pro-Inflammatory Cytokine Upregulation and Glial Reactivity After TBI

To characterize post-TBI inflammation, cortical transcript levels of pro-inflammatory cytokines were quantified via qRT-PCR. The results indicated a significant increase in IL-6, IL-1β, and TNF-α levels, alongside a decrease in IL-10 levels within the cortex post-TBI (Figure 2A–D). Furthermore, Immunofluorescence analyses detected substantially increased GFAP and IBA‑1 signals in the injured cortex (Figure 2E–H). Morphological examination indicated hypertrophy of astrocytic processes and activation of microglia, accompanied by an increased density of these glial cell types in comparison to sham tissue.

Figure 2 Cortical inflammatory responses after TBI. The relative mRNA expression levels of IL-6 (A), TNF-α (B), IL-1β (C), and IL-10 (D) among the Sham and TBI group on the third day post-injury (n = 6). (E) Immunofluorescence staining of IBA1 (red, microglia marker) and DAPI (blue) in mice on the third day (scale bar = 50 μm). (F) Immunofluorescence staining of GFAP (red, astrocyte marker) and DAPI (blue) in mice on the third day (scale bar = 50 μm). Quantification of immunofluorescence intensity for IBA1 expression is shown in (G), while (H) presents quantification for GFAP expression (n = 4). Data are expressed as mean ± SEM, with P values indicating comparisons to those of the TBI group.Data are expressed as the mean ± SEM.*** p <0.001.

Cortical snoRNA Regulation Post-TBI in Mice

In order to examine potential role of short nucleolar RNAs (snoRNAs) in neuropathology subsequent to TBI, we assessed cortical tissue samples from five TBI and five sham-operated control groups for snoRNA expression profiles utilizing a high-throughput small RNA microarray. We utilized screening criteria of p-value < 0.05 and |fold-change| ≥ 1.2 to analyze sequencing data. A total of 47 snoRNAs were identified through differential expression analysis across experimental groups (Table S2), revealing group-specific expression changes. Among these, 4 snoRNAs were found to be upregulated and 43 downregulated in TBI samples (Figure 3A and B). From this cohort of differentially expressed snoRNAs, we prioritized 12 candidates for downstream validation based on their statistically significant fold-changes or notably high baseline expression levels. However, only three snoRNAs—Gm24310, Gm22439, and Gm24418—demonstrated expression patterns that aligned with the initial sequencing results (Figure 3B and C). Notably, Gm24418 exhibited the most pronounced differential expression between the TBI and sham groups.

Figure 3 Cortical SnoRNA Expression Dynamics in CCI vs. Sham Groups Mice. (A) Volcano plot illustrating snoRNA expression profiles (p-value < 0.05, |fold-change| ≥ 1.2). (B) Heatmap showing hierarchical clustering of the expression levels of selected snoRNAs, (C) Quantitative expression validation of 12 selected snoRNAs using RT-PCR, Data are expressed as the mean ± SEM.* p <0.05;** p <0.01.

Characterization of Gm24418 in TBI Model Mouse

The FISH analysis of Gm24418 expression levels in different cell types, such as astrocytes, microglia, and neurons, confirmed significant enrichment in neurons (Figure 4A–C). Gm24418 was predominantly localized within the cytoplasm and nucleus. Collectively, these findings indicate that Gm24418 is an abundant and stable snoRNA that is differentially down-regulated in mouse cortical neurons following TBI. Similarly, this conclusion can be corroborated at the cellular level.

Figure 4 FISH analysis of Gm24418 expression in various brain cell types. (A) Gm24418 (red) shows significant localization in neurons, identified by NeuN staining (green), with reduced expression in TBI samples compared to sham. A quantitative analysis was conducted on cells that were double-labeled with Gm24418 and NeuN. (B) In microglia, marked by Iba-1 (green), and (C) astrocytes, identified by GFAP (green), Gm24418 exhibits an inconsistent presence across sham and TBI conditions. The scale bar represents 50 µm.Data are expressed as the mean ± SEM.** p <0.01.

Gm24418 Overexpression Potentiates LPS-Induced Neuronal Inflammation

Consistent with our earlier research, secondary neural degeneration following TBI appears to be substantially modulated by inflammatory cues originating from neurons. Therefore, we examined if Gm24418 modulates inflammatory signals in neurons. To elucidate this mechanism, N₂A cells were stimulated with the pro-inflammatory agent lipopolysaccharide (LPS), and treatment with 500 ng/mL LPS for twelve hours resulted in a significant reduction in Gm24418 mRNA levels (Figure 5A), suggesting its potential involvement in neuroinflammatory signaling pathways. Furthermore, overexpression of Gm24418 led to a marked decrease in the expression of key pro-inflammatory cytokines IL-6, TNF-α, and IL-1β in cultured N₂A cells (Figure 5B–E). These findings collectively support the hypothesis that Gm24418 plays a regulatory role in neuronal inflammatory responses following TBI.

Figure 5 Gm24418 expression in N2A subjected to LPS treatment. (A) Immunofluorescence analysis shows a marked increase in Gm24418-positive cells (red) in the LPS-treated N2A cells compared to sham, with nuclear counterstaining by DAPI (blue) confirming cell presence (scale bar = 100 μm). (B) Quantification of Gm24418-positive and DAPI-positive cells reveal a significant increase in LPS-treated samples, indicative of an inflammatory response. (C–E) Serum cytokine levels measured post-treatment highlight heightened expression of IL-6 (C), TNF-α (D), and IL-1β (E) in LPS-treated mice, significantly reduced upon co-treatment with Gm24418, suggesting an anti-inflammatory role for Gm24418. Data are expressed as the mean ± SEM.***p < 0.001.

The Gm24418 Interacts with CCL2 Protein in N₂A

We subsequently investigated the potential molecular mechanisms underlying Gm24418-induced inflammatory signaling in N₂A cells. Utilizing transcriptome sequencing, we demonstrated the differential expression of Gm24418 in neurons at the transcriptomic level. The analysis indicated that 125 genes were significantly upregulated, while 159 genes exhibited significant downregulation in the experimental LPS+Gm24418-mimics group compared to those in the LPS+NC mimics group, as opposed to comparisons between the those in the LPS+NC mimics and NC N₂A groups (Figure 6A and B). Notably, through analysis using the STRING database and CNV frequency analysis, CCL2 (C-C motif chemokine ligand 2) was identified as a candidate target gene (Figures 6C and S1). Fluorescence in situ hybridization revealed the effective co-localization of Gm24418 and Ccl2 (Figure 6D).

Figure 6 Integrated analysis of gene expression and interaction networks. (A) Venn diagram showing the overlapping DEGs identified in two comparison groups. (B) The volcano plot analysis identified CCL2 as a significantly differentially expressed gene. (C) Protein–protein interaction (PPI) network constructed from DEGs, with node reflecting centrality (larger nodes indicate higher centrality, representing hub genes), highlighting potential core regulatory molecules. (D) Binding sites of Gm24418 with Ccl2-WT and Ccl2-MUT. Statistical analysis of dual luciferase reporter gene assay confirmed the direct binding of Gm24418 to Ccl2.Data are expressed as the mean ± SEM.* p <0.05.

GO and KEGG Enrichment Profiling

The functional implications of the differentially expressed mRNAs were subsequently characterized through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, utilizing the bioinformatics platform Metascape. The GO enrichment analysis demonstrated targets were linked to biological activities including transcription cis-regulatory region binding, chemokine activity, and membrane-bounded organelle function. The KEGG pathway analysis demonstrated notable enrichment in pathways such as the TNF signaling pathway, MAPK signaling pathway, cytokine-cytokine receptor interaction, and NF-kB signaling pathway (Figure 7A and B). The findings from the GO biofunctional analysis indicate that CCL2 is key to synaptic plasticity, neuron projection extension, and synaptic signaling (FigureS2). These data indicate that CCL2 is predominantly implicated in signaling pathways associated with inflammatory responses. To further examine the regulatory influence of Gm24418 on CCL2 expression, qRT-PCR revealed CCL2 and TNF-α were highly elevated in neurons exposed to LPS-induced inflammatory responses; however, the overexpression of Gm24418 substantially mitigated this upregulation (Figure 7C and D).

Figure 7 The exploration of molecular mechanisms associated with Gm24418. (A) KEGG analysis identifying significantly enriched signaling pathways (with the TNF signaling pathway highlighted); (B) Schematic representation of the TNF signaling pathway depicting key molecular interactions; (C and D) qPCR results revealed relative mRNA expression levels of CCL2 and TNF-α in the Sham, NC and Gm24418-mimics groups, *p < 0.05;***p < 0.001.

Gm24418 Regulated Ccl2/TNF-α Signaling Pathway to Exert an Anti-Inflammation Effect

To evaluate the in vivo relevance of these findings, we conducted AAV-Gm24418 transfections prior to performing controlled cortical impact (CCI), as depicted in Figure 8A. Our results demonstrated a significant decrease in Gm24418 mRNA levels (Figure 8B and C) and an increase in pro-inflammatory cytokine levels (Figure 8D and E) in the tissues following the trauma. Notably, the overexpression of Gm24418 was able to reverse these changes. Similar alterations were observed in Ccl2 and TNF-α levels (Figure 8F and G). These findings confirm that Gm24418 exerts anti-inflammatory effects and facilitates functional recovery through the modulation of Ccl2, both in vitro and in vivo.

Figure 8 Gm24418 regulated Ccl2/TNF-α signaling pathway to exert an anti-inflammation effect. (A) Flowchart of in vivo animal experiment; (B and C) The expression of Gm24418 diminishes following CCI; The overexpression of Gm24418 can be mitigated through the preemptive administration of the AAV virus. A quantitative analysis was conducted on cells that tested positive for Gm24418; (D–G) qPCR results revealed relative mRNA expression levels of IL6, IL-1β, Ccl2 and TNF-α in the Sham, CCI and pAAV+CCI groups, Data are expressed as the mean ± SEM.* p <0.05;** p <0.01.

Discussion

Our investigation revealed that Gm24418, a small nucleolar RNA (snoRNA), exhibited dysregulated expression patterns in a microarray-based profiling study of TBI mouse models. Functional assays demonstrated that the forced expression of Gm24418 significantly suppressed neuroinflammatory responses in vivo, as evidenced by reduced microglial activation and decreased systemic levels of pro-inflammatory mediators, including IL-1β and TNF-α. Although snoRNAs have traditionally been characterized as molecular guides for ribosomal RNA (rRNA) modification,^32,33 Recent studies indicate that these non-coding RNAs have the ability to translocate to the cytoplasm, where they regulate mRNA stability and translation under stress conditions. They are also involved in post-transcriptional regulatory networks through sequence-specific interactions with mRNA transcripts, thereby affecting translational efficiency and mRNA stability.^31,34

Accumulating evidence stressed the critical regulatory roles of snoRNAs in the pathophysiological mechanisms underlying traumatic TBI. For instance, studies have demonstrated that snoRNA U13 modulates neuronal apoptosis by targeting caspase-3 activation,³⁵ thereby mitigating secondary brain damage in TBI models. The stability and relative abundance of snoRNAs in biofluids, enhanced by chemical modifications such as 2′-O-methylation, strengthen their potential as biomarkers for TBI.^36,37 Unlike conventional protein biomarkers (eg, GFAP, neuron-specific enolase), snoRNAs exhibit resistance to degradation and maintain stable expression in extracellular compartments, enabling sensitive detection in serum/plasma.³⁸ Recent studies have identified TBI-associated snoRNA signatures (eg, SNORD33, SNORA73) with diagnostic accuracy comparable to the established markers like GFAP.^39–41 The exploration of snoRNAs as diagnostic or prognostic biomarkers for TBI represents a promising research direction, given their specific expression changes and roles in post-transcriptional regulation.⁴⁰

Our transcriptome sequencing revealed that Gm24418 directly targets CCL2, a key chemokine involved in driving leukocyte infiltration during neuroinflammation.⁴¹ The CCL2 is implicated in the inflammatory response post-TBI.^42,43 Gm24418 regulates the neuronal secretome. By controlling the expression of genes such as Ccl2 and Il-6 within neurons, Gm24418 may influence the release of these and other signaling molecules into the extracellular space. These neuron-derived factors can then act on local glial cells, either promoting or suppressing their pro-inflammatory responses. It is connected to the recruitment of microglia and macrophages and facilitating the progression of phosphorylated tau pathology in the setting of repetitive TBI.⁴⁴ Furthermore, CCL2 is among the inflammatory mediators whose production can be stimulated by substance P, which potentiates neuroinflammation following TBI.^45,46 Specifically, the interaction with Gm24418 to the 3′-UTR of CCL2 suppressed its expression, consequently inhibiting monocyte recruitment to sites of injury. This data corroborates other reports suggesting that the reduction of CCL2 alleviates inflammation and neuronal damage caused by TBI.^47,48

Moreover, Gm24418 likely affects multiple targets. While CCL2 was the most significant direct target identified via bioinformatics, the downregulation of the TNF signaling pathway represents a systemic cellular response, a crucial regulator of the NF-κB and MAPK cascades, and this attenuation leads to decreased nuclear translocation of p65 and lower phosphorylation levels of IκB-α.⁴⁹ The TNF-α signaling pathway has a multifaceted involvement in pathogenesis of TBI.⁵⁰ Acute TBI triggers pro-inflammatory microglial polarization and TNF receptor 1 (TNF-R1) upregulation, driving pro-inflammatory cytokine release (eg, TNF-α) while depleting anti-inflammatory mediators like IL-10,⁵¹ which contribute to neuroinhibition, neuronal death, and organ dysfunction.⁵² TNF-α has been reported to exhibit both neuroprotective and neurotoxic effects, which may depend on the mode, method, and cellular source.⁵³ Notably, pharmacological inhibition of NF-κB—a downstream effector of TNF signaling,^54,55 has been shown to alleviate TBI-induced damage. Specifically, blocking NF-κB signaling with the specific inhibitor SN50 reduced neural cell plasma membrane integrity disruption, decreased brain tissue lesion volume, and improved motor and learning memory functions after TBI.⁵⁶ This was associated with decreased expression of apoptosis-related proteins (eg, TNF-α, caspase-3, tBid),^57,58 autophagy/lysosome pathway-related proteins (eg, Cathepsin B),^59,60 and inflammation-related factors (eg, TNF-α, caspase-1),^61,62 and increased expression of the neural regeneration marker Nestin.^63,64 Notably, inhibition of TNF has been demonstrated to alleviate BBB disruption while enhancing functional recovery following TBI. Our results suggest that Gm24418’s attenuation of neuroinflammation and its protective effects in TBI may partly occur through the modulation of the TNF signaling pathway.

Several limitations exist in this study. First, the spatial distribution of Gm24418 in cell types beyond the neuronal populations examined remains unclear. While our findings confirm its presence in NeuN-positive neurons within the perilesional area and indicate a significant increase in cultured neurons subjected to LPS stimulation, it is plausible that Gm24418 also plays a role in modulating inflammatory pathways within astrocytes and microglia. Testing this hypothesis will be a critical objective for future experiments. Second, one important consideration when interpreting the reduced Gm24418 expression in the TBI group is the potential contribution of focal tissue loss. While we carefully sampled the perilesional cortex to avoid the necrotic core, we cannot completely rule out the possibility that some of the observed reduction is due to a loss of Gm24418 expressing cells. Nevertheless, future studies utilizing in situ hybridization or single-cell RNA sequencing would be valuable to definitively distinguish between cell loss and transcriptional downregulation at the single-cell level. Third, overexpression of Gm24418 did not suppress all chemokines elevated following traumatic brain injury (TBI) or LPS exposure, suggesting that additional inflammatory mediators may interact with or be influenced by Gm24418. Fourth, although using LPS as an in vitro stimulus does not fully capture the complexity of TBI-specific pathophysiology, it is currently regarded as a robust proof-of-concept approach for mechanistic studies. Collectively, further investigations are necessary to evaluate the potential of Gm24418 as a viable target for clinical therapeutic strategies.

Conclusion

The snoRNA Gm24418 was abundantly expressed in mouse brain neurons and exhibited marked downregulation following TBI. It functions as an anti-inflammatory snoRNA during TBI through dual regulation of CCL2-dependent leukocyte chemotaxis and the TNF signaling pathway. These findings position snoRNAs as promising targets for anti-inflammatory strategies in managing TBI.